Vulnerable periods for the mouse mammary gland: comparison of the effects of ethinyl estradiol exposures during two early stages of development.

The mammary gland is responsive to endogenous hormones and environmental chemicals that are estrogen receptor (ER) agonists. The mouse mammary gland offers the opportunity to dissect the most sensitive windows of exposure. 17α-ethinyl estradiol (EE2) is a pharmaceutical ER agonist that often serves as a positive control for estrogen-active chemicals. Here, adult female mice were exposed to EE2 starting either at pregnancy day 7, or on lactational day 1, and exposures continued until the litters were weaned. The pups were therefore exposed during gestation + the juvenile period, or during the juvenile period alone. The morphology of the mammary gland was evaluated in both male and female offspring at two life stages: weaning (postnatal day [PND]21) and at puberty (PND32). Other hormone-sensitive outcomes evaluated included body weight, anogenital index, frequency of open vagina, and weight of the uterus. We found age- and sex-dependent effects of EE2 on these estrogen-responsive endpoints including the morphology of the mammary gland. Importantly, EE2 altered mammary gland morphology even when exposures were limited to the juvenile period. However, the number of endpoints that were affected in animals from the EE2-Juvenile-Only period were fewer, and typically of a lower magnitude, compared to those observed in the EE2-Gest-Juvenile group. Understanding the effects of environmental estrogen exposures during the juvenile period is critical because humans are exposed to estrogenic pollutants throughout life, including in early childhood.

The Dual Faces of Oestrogen: The Impact of Exogenous Oestrogen on the Physiological and Pathophysiological Functions of Tissues and Organs.

Oestrogen plays a crucial physiological role in both women and men. It regulates reproductive functions and maintains various non-reproductive tissues through its receptors, such as oestrogen receptor 1/oestrogen receptor α (ESR1/Erα), oestrogen receptor 2/oestrogen receptor ß (ESR2/Erß), and G protein-coupled oestrogen receptor 1 (GPER). This hormone is essential for the proper functioning of women's ovaries and uterus. Oestrogen supports testicular function and spermatogenesis in men and contributes to bone density, cardiovascular health, and metabolic processes in both sexes. Nuclear receptors Er-α and Er-ß belong to the group of transcription activators that stimulate cell proliferation. In the environment, compounds similar in structure to the oestrogens compete with endogenous hormones for binding sites to receptors and to disrupt homeostasis. The lack of balance in oestrogen levels can lead to infertility, cancer, immunological disorders, and other conditions. Exogenous endocrine-active compounds, such as bisphenol A (BPA), phthalates, and organic phosphoric acid esters, can disrupt signalling pathways responsible for cell division and apoptosis processes. The metabolism of oestrogen and its structurally similar compounds can produce carcinogenic substances. It can also stimulate the growth of cancer cells by regulating genes crucial for cell proliferation and cell cycle progression, with long-term elevated levels linked to hormone-dependent cancers such as breast cancer. Oestrogens can also affect markers of immunological activation and contribute to the development of autoimmune diseases. Hormone replacement therapy, oral contraception, in vitro fertilisation stimulation, and hormonal stimulation of transgender people can increase the risk of breast cancer. Cortisol, similar in structure to oestrogen, can serve as a biomarker associated with the risk of developing breast cancer. The aim of this review is to analyse the sources of oestrogens and their effects on the endogenous and exogenous process of homeostasis.

Methylimidazolium ionic liquids - A new class of forever chemicals with endocrine disrupting potential.

A class of chemical with a potentially important perceived future contribution to the net zero carbon goal (as "green" solvents) is the methylimidazolium ionic liquids (MILs). These solvents are used in industrial processes such as biofuel production yet little is known about their environmental stability or toxicity in man although one MIL - 1-octyl-3-methylimidazolium (M8OI) - has been shown to activate the human estrogen receptor alpha (ERα). The stabilities of the chloride unsubstituted methylimidazolium (MI) and MILs possessing increasing alkyl chain lengths (2C, 1-ethyl-3-methylimidazolium (EMI); 4C, 1-butyl-3-methylimidazolium (BMI); 6C; 1-hexyl-3-methylimidazolium (HMI), 8C, M8OI; 10C, 1-decyl-3-methylimidazolium (DMI)) were examined in river water and a human liver model system. The MILs were also screened for their abilities to activate the human ERα in vitro and induce uterine growth in pre-pubertal rats in vivo. Short chain MILs (EMI, BMI and HMI) underwent negligible metabolism and mineralisation in river water; were not metabolised in a model of human liver metabolism; activated the human ERα in vitro and were estrogenic in vivo in rats. A structure-based computational approach predicted short chain MIL binding to both the estrogen binding site and an additional site on the human estrogen receptor alpha. Longer chain MILs (M8OI and DMI) were metabolised in river water and partially mineralised. Based on structure-activity considerations, some of these environmentally-derived metabolites may however, remain a hazard to the population. MILs therefore have the potential to become forever chemicals with adverse effects to both man, other animals and the environment in general.

Multiple xenoestrogen air pollutants and breast cancer risk: Statistical approaches to investigate combined exposures effect.

Studies suggested that exposure to air pollutants, with endocrine disrupting (ED) properties, have a key role in breast cancer (BC) development. Although the population is exposed simultaneously to a mixture of multiple pollutants and ED pollutants may act via common biological mechanisms leading to synergic effects, epidemiological studies generally evaluate the effect of each pollutant separately. We aimed to assess the complex effect of exposure to a mixture of four xenoestrogen air pollutants (benzo-[a]-pyrene (BaP), cadmium, dioxin (2,3,7,8-Tétrachlorodibenzo-p-dioxin TCDD)), and polychlorinated biphenyl 153 (PCB153)) on the risk of BC, using three recent statistical methods, namely weighted quantile sum (WQS), quantile g-computation (QGC) and Bayesian kernel machine regression (BKMR). The study was conducted on 5222 cases and 5222 matched controls nested within the French prospective E3N cohort initiated in 1990. Annual average exposure estimates to the pollutants were assessed using a chemistry transport model, at the participants' residence address between 1990 and 2011. We found a positive association between the WQS index of the joint effect and the risk of overall BC (adjusted odds ratio (OR) = 1.10, 95% confidence intervals (CI): 1.03-1.19). Similar results were found for QGC (OR = 1.11, 95%CI: 1.03-1.19). Despite the association did not reach statistical significance in the BKMR model, we observed an increasing trend between the joint effect of the four pollutants and the risk of BC, when fixing other chemicals at their median concentrations. BaP, cadmium and PCB153 also showed positive trends in the multi-pollutant mixture, while dioxin showed a modest inverse trend. Despite we found a clear evidence of a positive association between the joint exposure to pollutants and BC risk only from WQS and QGC regression, we observed a similar suggestive trend using BKMR. This study makes a major contribution to the understanding of the joint effects of air pollution.

Oestrogens, adipose tissues and environmental exposures influence obesity and diabetes across the lifecycle.

Endogenous oestrogens regulate essential functions to include menstrual cycles, energy balance, adipose tissue distribution, pancreatic ß-cell function, insulin sensitivity and lipid homeostasis. Oestrogens are a family of hormones which include oestradiol (E2), oestrone (E1) and oestriol (E3). Oestrogens function by binding and activating oestrogen receptors (ERs). Phytoestrogens are plant-derived compounds which exhibit oestrogenic-like activity and can bind to ERs. Phytoestrogens exert potential oestrogenic-like benefits; however, their effects are context-dependent and require cautious consideration regarding generalised health benefits. Xenoestrogens are synthetic compounds which have been determined to disrupt endocrine function through binding to ERs. Xenoestrogens enter the body through various routes and given their chemical structure they can accumulate, posing long-term health risks. Xenoestrogens interfere with endogenous oestrogens and their functions contributing to conditions like cancer, infertility, and metabolic disorders. Understanding the interplay between endogenous and exogenous oestrogens is critical in order to determine their potential health consequences and requires further investigation. This manuscript provides a summary of the role endogenous oestrogens have in regulating metabolic functions. Additionally, we discuss the impact phytoestrogens and synthetic xenoestrogens have on biological systems across various life stages. We highlight their mechanisms of action, potential benefits, risks and discuss the need for further research to bridge gaps in understanding and mitigate exposure-related health risks.

A mechanistic toxicology study to grasp the mechanics of zearalenone estrogenicity: Spotlighting aromatase and the effects of its genetic variability.

Zearalenone (ZEN) is a mycoestrogen produced by Fusarium fungi contaminating cereals and in grain-based products threatening human and animal health due to its endocrine disrupting effects. Germane to the mechanisms of action, ZEN may activate the estrogen receptors and inhibit the estrogens-producing enzyme aromatase (CYP19A1). Both show single nucleotide variants (SNVs) among humans associated with a diverse susceptibility of being activated or inhibited. These variations might modify the endocrine disrupting action of ZEN, requiring dedicated studies to improve its toxicological understanding. This work focused on human aromatase investigating via 3D molecular modelling whether some of the SNVs reported so far (n = 434) may affect the inhibitory potential of ZEN. It has been also calculated the inhibition capability of α-zearalenol, the most prominent and estrogenically potent phase I metabolite of ZEN, toward those aromatase variants with an expected diverse sensitivity of being inhibited by ZEN. The study: i) described SNVs likely associated with a different susceptibility to ZEN and α-zearalenol inhibition - like T310S that is likely more susceptible to inhibition, or D309G and S478F that are possibly inactive variants; ii) proofed the possible existence of inter-individual susceptibility to ZEN; iii) prioritized aromatase variants for future investigations toward a better comprehension of ZEN xenoestrogenicity at an individual level.

Effects of butyl benzyl phthalate exposure during pregnancy and lactation on the post-involution mammary gland.

The mammary gland undergoes comprehensive reorganization during pregnancy, lactation, and subsequent involution. Following involution, the mammary gland has structural and functional differences compared to the gland of a nulliparous female. These parity-associated changes are regulated by hormones and may be vulnerable to endocrine-disrupting chemicals (EDCs). In this study, we evaluated the long-term effects of butyl benzyl phthalate (BBP), an estrogenic plasticizer, on the parous mouse mammary gland. Pregnant BALB/c mice were treated with 0, 3, 500, or 18000 µg/kg/day BBP throughout both pregnancy and the lactational period. The litters born to these females were evaluated for litter size and growth. The parous females were then kept for five weeks following weaning of the pups, during which period there was no exposure to BBP. After five weeks of post-weaning, mammary glands were collected and assessed for changes in histomorphology, steroid receptor expression, innate immune cell number, and gene expression. An unexposed age-matched nulliparous control was also evaluated as a comparator group. BBP increased male and female pup weight at puberty and female offspring in adulthood. BBP also altered innate immune cells in the post-involution mammary gland, reducing the effect of parity on macrophages. Lastly, BBP modestly increased mammary gland ductal complexity and periductal structure, but had no effect on expression of estrogen receptor, progesterone receptor, or a marker of proliferation. These results suggest that BBP may interfere with some effects of parity on the mouse mammary gland and induce weight gain in exposed offspring.

Estrogens and xenoestrogen residues in manure-based fertilizers and their potential ecological risks.

Optimal manure treatment aimed at usage as agricultural soil fertilizers is a prerequisite ecological pollution control strategy. In this work, livestock manure-based fertilizers were collected from 71 animal farms across 14 provinces in China. The contamination levels and potential ecotoxicological risks of residual steroid estrogens (SEs): estrone (E1), estriol (E3), 17α-estradiol (17α-E2), 17ß-estradiol (17ß-E2) and xenoestrogen (XE) bisphenol A (BPA), were investigated. The results showed that the occurrence frequencies for SEs and XE ranged from 66.67% to 100%, and the mean concentration varied considerably across the study locations. The total content of SEs and XE in Hebei province was the highest, and swine manure-based fertilizers concentrations were higher than the levels reported in other animal fertilizers. Compared with farm level manure, manure-based fertilizers are processed by composting, and the micropollutants quantities are significantly reduced (mean: 87.65 - 534.02 µg/kg). The total estradiol equivalent quantity (EEQ) that might migrate to the soil was estimated to be 1.23 µg/kg. Based on the estimated application rate of manure, 38% of the fertilizers risk quotients exceeded 0.1, indicating medium to high risks pressure on terrestrial organisms. Nonetheless, the estrogenic risk was lower in manure-based fertilizers than in manure. This study highlights the significance of proper treatment of livestock manure and designing an optimal manure fertilization strategy to mitigate the risks posed by SEs and XEs to the agroecosystems.

Ex vivo effects of bisphenol A or zearalenone on the prepubertal rat testis.

Bisphenol A (BPA) and zearalenone (ZEA) are two widespread xenoestrogens involved in male reproductive disorders. Few studies investigated the effects of these compounds on the prepubertal testis, which is highly sensitive to endocrine disruptors such as xenoestrogens. An ex vivo approach was performed to evaluate the effects of BPA or ZEA (10-11, 10-9, 10-6 M) on the testes of 20 and 25 dpp rats. To investigate the involvement of classical nuclear ER-mediated estrogen signaling in these effects, pre-incubation with an antagonist (ICI 182.780 10-6 M) was performed. BPA and ZEA have similar effects on spermatogenesis- and steroidogenesis-related endpoints in the immature testis, but our study highlights different age-dependent patterns of sensitivity to each compound during the prepubertal period. Moreover, our results indicate that the effects of BPA are likely to be induced by nuclear ER, whereas those of ZEA appear to involve other mechanisms.

The effect of long-term exposure to nonylphenol at environmentally relevant levels on mouse liver and adipose tissue.

Exposure to the xenoestrogen nonylphenol (NP) during critical windows of development leads to metabolic abnormalities in adult life. However, less is known about NP exposure outside the developmental period on metabolic outcomes. We investigated the effect of prolonged exposure to NP after sexual maturity and at environmentally relevant concentrations below the 'no observable adverse effects level' (0.5 and 2.5 mg/kg/d). Male Swiss mice fed a normal-fat diet exposed to 2.5 mg/kg/d NP showed reduced weight gain and hepatic fat content. In male and female C57BL/6 mice fed a high-fat diet, NP exposure modified the mRNA levels of estrogen receptor α (Esr1) and adipose lineage markers in a sexually dimorphic and adipose depot-dependent pattern. Moreover, in primary female but not male stromal vascular cells from C57BL/6 mouse inguinal WAT induced to differentiate into adipocytes, NP upregulated Fabp4 expression. Low-level exposure to NP outside critical developmental windows may affect the metabolic phenotype distinctly. DATA AVAILABILITY STATEMENT: All data not included in the manuscript, such as raw results, are available upon request and should be addressed to AAA.

The impacts of exposure to bisphenol A in the adult female prostate Meriones unguiculatus.

The female prostate is associated with the urogenital system and presents homology in morphological terms with the male prostate. Due to its responsiveness to endogenous hormones, this gland is under a constant risk of developing prostatic pathologies and neoplasia when exposed to certain exogenous compounds. Bisphenol A (BPA) is an endocrine disruptor found in different plastic and resin products. Studies have emphasized the effects of perinatal exposure to this compound on different hormone-responsive organs. However, there have been few studies highlighting the influence on female prostate morphology of perinatal exposure to BPA. The objective of this study was to describe the histopathological alterations caused by perinatal exposure to BPA (50 µg/kg) and 17-ß estradiol (E2) (35 µg/kg) in the prostate of adult female gerbils. The results showed that E2 and BPA induced proliferative lesions in the female prostate and acted along similar pathways by modulating steroid receptors in the epithelium. BPA was also found to be a pro-inflammatory and pro-angiogenic agent. The impacts of both agents were marked in the prostatic stroma. An increase in the thickness of the smooth muscle layer and a decrease in AR expression were observed, but no alterations in the expression of ERα and ERß, leading to estrogenic sensitivity of the prostate. However, a peculiar response of the female prostate was to diminish the collagen frequency under BPA exposure correlated to smooth muscle layer. These data therefore indicate the development of features related to estrogenic and non-estrogenic tissue repercussions by BPA perinatally exposure in gerbil female prostate.

Comparative Analysis of Transcriptomic Changes including mRNA and microRNA Expression Induced by the Xenoestrogens Zearalenone and Bisphenol A in Human Ovarian Cells.

Xenoestrogens are natural or synthetic compounds that mimic the effect of endogenous estrogens and might cause cancer. We aimed to compare the global transcriptomic response to zearalenone (ZEA; mycotoxin) and bisphenol A (BPA; plastic additive) with the effect of physiological estradiol (E2) in the PEO1 human ovarian cell line by mRNA and microRNA sequencing. Estrogen exposure induced remarkable transcriptomic changes: 308, 288 and 63 genes were upregulated (log2FC > 1); 292, 260 and 45 genes were downregulated (log2FC < -1) in response to E2 (10 nM), ZEA (10 nM) and BPA (100 nM), respectively. Furthermore, the expression of 13, 11 and 10 miRNAs changed significantly (log2FC > 1, or log2FC < -1) after exposure to E2, ZEA and BPA, respectively. Functional enrichment analysis of the significantly differentially expressed genes and miRNAs revealed several pathways related to the regulation of cell proliferation and migration. The effect of E2 and ZEA was highly comparable: 407 genes were coregulated by these molecules. We could identify 83 genes that were regulated by all three treatments that might have a significant role in the estrogen response of ovarian cells. Furthermore, the downregulation of several miRNAs (miR-501-5p, let-7a-2-3p, miR-26a-2-3p, miR-197-5p and miR-582-3p) was confirmed by qPCR, which might support the proliferative effect of estrogens in ovarian cells.

Estrogen Signaling Influences Nephron Segmentation of the Zebrafish Embryonic Kidney.

Despite significant advances in understanding nephron segment patterning, many questions remain about the underlying genes and signaling pathways that orchestrate renal progenitor cell fate choices and regulate differentiation. In an effort to identify elusive regulators of nephron segmentation, our lab conducted a high-throughput drug screen using a bioactive chemical library and developing zebrafish, which are a conserved vertebrate model and particularly conducive to large-scale screening approaches. 17ß-estradiol (E2), which is the dominant form of estrogen in vertebrates, was a particularly interesting hit from this screen. E2 has been extensively studied in the context of gonad development, but roles for E2 in nephron development were unknown. Here, we report that exogenous estrogen treatments affect distal tubule composition, namely, causing an increase in the distal early segment and a decrease in the neighboring distal late. These changes were noted early in development but were not due to changes in cell dynamics. Interestingly, exposure to the xenoestrogens ethinylestradiol and genistein yielded the same changes in distal segments. Further, upon treatment with an estrogen receptor 2 (Esr2) antagonist, PHTPP, we observed the opposite phenotypes. Similarly, genetic deficiency of the Esr2 analog, esr2b, revealed phenotypes consistent with that of PHTPP treatment. Inhibition of E2 signaling also resulted in decreased expression of essential distal transcription factors, irx3b and its target irx1a. These data suggest that estrogenic compounds are essential for distal segment fate during nephrogenesis in the zebrafish pronephros and expand our fundamental understanding of hormone function during kidney organogenesis.

Potential of Biochar from Wood Gasification to Retain Endocrine Disrupting Chemicals.

In this study, a biochar obtained from poplar wood gasification at a temperature of 850 °C was used to adsorb the xenoestrogens 4-tert-octylphenol (OP) and bisphenol A (BPA) and the herbicide metribuzin from water. Scanning electron microscopy (SEM-EDX) and Fourier-transform infrared (FTIR) spectroscopy were employed to investigate the surface micromorphology and functional groups composition of biochar, respectively. The study of sorption kinetics showed that all compounds achieved the steady state in less than 2 h, according to a pseudo-second order model, which denoted the formation of strong bonds (chemisorption) between biochar and the compounds. Adsorption isotherms data were described by the Henry, Freundlich, Langmuir and Temkin equations. At temperatures of 10 and 30 °C, the equilibrium data of the compounds were generally better described by the Freundlich model, although, in some cases, high correlation coefficients (r ≥ 0.98) were obtained for more than one model. Freundlich constants, KF, for OP, BPA and metribuzin were, respectively, 218, 138 and 4 L g-1 at 10 °C and 295, 243 and 225 L g-1 at 30 °C, indicating a general increase of adsorption at higher temperature. Desorption of all compounds, especially OP and BPA, from biochar was slow and very scarce, denoting an irreversible and hysteretic process. Comparing the results of this study with those reported in the literature, we can conclude that the present biochar has a surprising ability to retain organic compounds almost permanently, thus behaving as an excellent low-cost biosorbent.

Microplastic in northern anchovies (Engraulis mordax) and common murres (Uria aalge) from the Monterey Bay, California USA - Insights into prevalence, composition, and estrogenic activity.

Microplastic (particle size <5 mm) is considered an emerging threat to the marine environment, yet data are limited for coastal ecosystems. To provide information related to microplastic in a coastal system, we used alkaline tissue digestion and Raman spectroscopy to quantify the prevalence and composition (e.g. fiber, fragment, foam, etc.) of anthropogenic microparticles in the digestive tracts of northern anchovies (Engraulis mordax, anchovy, n = 24), and common murres (Uria aalge, murre, n = 19) from the Monterey Bay, California USA. We also determined microplastic prevalence and composition in seawater (n = 12 17-h sampling periods representing ∼46,000 L sampled) from two Monterey Bay intake systems (Moss Landing, CA and Santa Cruz, CA USA). Microparticles recovered from murre digestive tracts were assessed for estrogenic activity using an in-vitro estrogen receptor activation assay. Suspected anthropogenic microparticles based on visual characteristics were recovered from all sample types with ∼2 particles per 1000 L from the seawater sampling periods, 58% prevalence in anchovies, and 100% prevalence in murres. Across samples of seawater, anchovies, and murres, the most abundant microparticle type found were fibers (78%), followed by fragments (13%), foam (6%), film (2%), and beads (1%). Raman spectroscopy identified 57% of microparticles (excluding dye-prominent and unknown) as plastic (synthetic, semi-synthetic, or blends). Almost one quarter (23%) of the murre digestive tracts contained microparticles that exhibited estrogenic activity. Our study describes the widespread occurrence, composition, and potential estrogenic activity of microplastic in the Monterey Bay and provides important information to aid in the understanding of microplastic contamination in coastal systems.

Bisphenol A-Induced Endocrine Dysfunction and its Associated Metabolic Disorders.

Bisphenol A (BPA) is an endocrine-disrupting chemical widely present in many consumer goods that poses a significant threat to our health upon exposure. Humans are exposed to BPA, which directly or indirectly causes endocrine dysfunctions that lead to metabolic disorders like obesity, fatty liver diseases, insulin resistance, polycystic ovarian syndrome, and other endocrine- related imbalances. The duration, quantity, and period of exposure to BPA, especially during the critical stage of development, determine its impact on reproductive and non-reproductive health. Because of its endocrine-disrupting effects, the European Chemical Agency has added BPA to the candidate list of chemicals of very serious concern. Due to its estrogenic properties and structural similarities with thyroid hormones, BPA disrupts the endocrine system at different levels. It interacts with estrogen receptors at the molecular level and acts as an antagonist or agonist via an estrogen receptor-dependent signaling pathway. In particular, BPA binds to G-protein coupled receptors and estrogen receptors, activating signaling pathways that influence cellular apoptosis, proliferation, differentiation, and inflammation. BPA acts as an obesogen that promotes adipogenesis and correlates with increased lipid accumulation and elevated expression of adipogenic markers. As a metabolic and endocrine disruptor, BPA impairs cellular homeostasis by increasing oxidative mediators and decreasing antioxidant enzymes, resulting in mitochondrial dysfunction. Due to its endocrine-disrupting properties, BPA exposure induces endocrine dysfunctions, causing metabolic syndrome. This review article gives recent development and novel insights into the cellular and molecular mechanisms of BPA-induced endocrine dysfunctions and their associated metabolic disorders.

In vitro evaluation for estrogenic mechanisms of the disinfectant benzalkonium chloride as an emerging contaminant

This study aimed to evaluate in vitro the possible mechanisms underlying the estrogenic potential of benzalkonium chloride (BAC) as a disinfectant emerging contaminant. Effects of BAC at the environmentally-relevant concentrations on estrogen synthesis and estrogen receptor (ER) signaling were assessed using the H295R steroidogenesis assay and the MCF-7 proliferation assay, respectively. Results showed that exposure to BAC at concentrations of 1.0-1.5 mg/L for 48 h significantly increased estradiol production of H295R cells in a concentration-dependent manner. Transcription of steroidogenic genes 3β‐HSD2, 17β‐HSD1, 17β‐HSD4, and CYP19A were significantly enhanced by BAC. In ER-positive MCF-7 cells, exposure to 0.5-1.5 mg/L BAC for 48 h significantly promoted cell proliferation and increased the expressions of ERα and G-protein coupled estrogen receptor 1. Flow cytometry analysis showed that 0.5-1.5 mg/L BAC significantly decreased the percentage of cells in G0/G1 phase, increased the percentage in S phase, and BAC at concentrations of 1.0 and 1.5 mg/L increased the G2/M phase cells. Findings of the study suggested that BAC at environmentally-relevant concentrations might act as a xenoestrogen through its inhibitory effect on steroidogenesis and ER-mediated mechanism.

Failure to Launch: The Endocrine Disruptor Screening Program at the U.S. Environmental Protection Agency.

It has been 25 years since the U.S. Congress passed the Food Quality Protection Act of 1996, an amendment to the Food Drug and Cosmetic Act, which mandated that the US Environmental Protection Agency (EPA) test all pesticide chemicals used in food for endocrine disruption. Soon after the law passed, EPA established the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to provide recommendations to the agency on how its Endocrine Disruptor Screening Program (EDSP) should work. Among them, the committee recommended that EDSP screening should 1) evaluate both human and ecological effects; 2) test for disruption of the estrogen, androgen, and thyroid systems; 3) evaluate pesticide and non-pesticide chemicals; and 4) implement a tiered approach. EPA adopted the recommendations and the EDSP was created in 1998. To date, the EPA has yet to fully implement the law; in other words, it has failed to test all pesticide chemicals for endocrine disruption. Of the small number that have been tested, not a single pesticide chemical has been determined to be an endocrine disruptor, and no regulatory actions have been taken. Here, we review the missed opportunities EPA had to make the EDSP a functional and effective program aimed at protecting human health and the environment. Two reports by the EPA's Office of Inspector General from 2011 to 2021 provide the framework for our discussion.

Exposure to Low Doses of Oxybenzone During Perinatal Development Alters Mammary Gland Stroma in Female Mice.

Mammary stroma is a prominent modulator of epithelial development, and a complex set of interactions between these tissue compartments is essential for normal development, which can be either permissive or restrictive in tumor initiation and progression. During perinatal development, exposures of mice to oxybenzone, a common UV filter, environmental pollutant and endocrine disruptor, induce alterations in mammary epithelium. Our prior research indicates that oxybenzone alters mammary epithelial structures at puberty and in adulthood. We had also previously observed changes in the expression of hormone receptors at puberty (e.g., oxybenzone induced a decrease in the number of epithelial cells positive for progesterone receptor) and in adulthood (e.g., oxybenzone induced a decrease in the number of estrogen receptor-positive epithelial cells), and increased body weight in adulthood. Here, we investigated mammary stromal changes in BALB/c animals exposed during gestation and perinatal development to 0, 30, or 3000 µg oxybenzone/kg/day. In mice exposed to 30 µg/kg/day, we observed morphological changes in adulthood (e.g., a thicker periductal stroma and adipocytes that were considerably larger). We also observed an increased number of mast cells in the mammary stroma at puberty which may represent a transient influence of oxybenzone exposure. These results provide additional evidence that even low doses of oxybenzone can disrupt hormone sensitive outcomes in the mammary gland when exposures occur during critical windows of development, and some of these effects manifest in later life.

Widespread alterations upon exposure to the estrogenic endocrine disruptor ethinyl estradiol in the liver proteome of the marine male fish Cyprinodon variegatus.

Quantitative proteomic changes in the liver of adult males of Sheepshead minnow (Cyprinodon variegatus) upon exposure to ethinyl estradiol (EE2) were assessed to provide an advanced understanding of the metabolic pathways affected by estrogenic endocrine disruption in marine fish, and to identify potential novel molecular biomarkers for the environmental exposure to estrogens. From a total of 3188 identified protein groups (hereafter proteins), 463 showed a statistically significant difference in their abundance between EE2 treatment and solvent control samples. The most affected biological processes upon EE2 exposure were related to ribosomal biogenesis, protein synthesis and transport of nascent proteins to endoplasmic reticulum, and nuclear mRNA catabolism. Within the group of upregulated proteins, a subset of 14 proteins, involved in egg production (Vitellogenin, Zona Pellucida), peptidase activity (Cathepsine E, peptidase S1, Serine/threonine-protein kinase PRP4 homolog, Isoaspartyl peptidase and Whey acidic protein), and nucleic acid binding (Poly [ADP-ribose] polymerase 14) were significantly upregulated with fold-change values higher than 3. In contrast, Collagen alpha-2, involved in the process of response to steroid hormones, among others, was significantly downregulated (fold change = 0.2). This pattern of alterations in the liver proteome of adult males of C. variegatus can be used to identify promising novel biomarkers for the characterization of exposure of marine fish to estrogens. The Whey acidic protein-like showed the highest upregulation in EE2-exposed individuals (21-fold over controls), suggesting the utility of abundance levels of this protein in male liver as a novel biomarker of xenoestrogen exposure.